Employment of a Newly Defined In Vitro Fertilization Protocol to Determine the Cytoskeletal Machinery, DNA Damage, and Subsequent DNA Repair Resulting from Endocrine Disruption by Hexavalent Chromium in Rat Metaphase II Oocytes.

Abstract

These protocols describe a detailed method to determine the DNA damage and F-actin and microtubule defects of metaphase II oocytes caused by hexavalent chromium, Cr(VI), an endocrine disrupting chemical (EDC). The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing in vitro fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F-actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early-stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F-actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC-mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F-actin), and assess expression of the DNA double-strand break marker γ-H2AX and the DNA repair protein RAD51 in control and Cr(VI)-exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. © 2024 Wiley Periodicals LLC. Basic Protocol 1: In vivo EDC treatment of rats and immunostaining of treated oocytes Basic Protocol 2: In vitro fertilization and immunostaining of early-stage embryos.